Xenopus laevis control the change from no DNA synthesis in sperm and egg nuclei to extremely rapid nuclear DNA replication in the fertilized egg and early embryo. To discover what these components are and how they exert their control is the specific objective of this project. The significance of this research lies not only in the essential role of DNA replication in growth and development, but also in the fact that regional differences in DNA synthesis characteristically are one of the first indications of pattern formation (with subsequent cellular differentiation) during embryogenesis. An efficient cell-free assay for initiation of DNA replication in isolated nuclei or in purified polyoma DNA molecules by cytoplasmic components from eggs or early embryos of Xenopus laevis has been developed by the principal investigator. DNA replication eyes appear in vitro during incubation parallelling those appearing in vivo during embryogenesis; both types will be studied. The cytoplasmic components, several of which are found only at specific developmental stages, can be separated by column chromatography into at least 9 fractions required for DNA replication in vitro: these components will be characterized. The localization within the oocyte or embryo of each component and its time of synthesis will be investigated. The key technique underlying this project is the identification by electron microscopy of intermediates in DNA replication generated in vitro. Coupled with standard purification and isotype incorporation, procedures of nucleic acid enzymology, this assay provides an extraordinarily powerful way to identify the factors controlling DNA replication in early embryos. A unique advantage of Xenopus laevis is that as each component is identified and purified, its role in vivo in the control of DNA synthesis can be assessed by microinjection.